One-Step, Multiplexed Fluorescence Detection of microRNAs Based on Duplex-Specific Nuclease Signal Amplification

TLDR

Traditional molecular beacons suffer from low sensitivity because each target molecule activates only one fluorescent probe. The authors aim to develop a duplex‑specific nuclease signal amplification (DSNSA) strategy to enhance beacon sensitivity and enable rapid, multiplex quantification of miRNAs. DSNSA uses DSN to repeatedly cleave TaqMan probes in the presence of a target, allowing one miRNA to generate fluorescence from thousands of probes. The method achieves femtomolar detection limits—about five orders of magnitude better than conventional beacons—while discriminating closely related miRNA family members, making it a promising tool for routine multiplex miRNA analysis in tissues and cells.

Abstract

Traditional molecular beacons, widely applied for detection of nucleic acids, have an intrinsic limitation on sensitivity, as one target molecule converts only one beacon molecule to its fluorescent form. Herein, we take advantage of the duplex-specific nuclease (DSN) to create a new signal-amplifying mechanism, duplex-specific nuclease signal amplification (DSNSA), to increase the detection sensitivity of molecular beacons (Taqman probes). DSN nuclease is employed to recycle the process of target-assisted digestion of Taqman probes, thus, resulting in a significant fluorescence signal amplification through which one target molecule cleaves thousands of probe molecules. We further demonstrate the efficiency of this DSNSA strategy for rapid direct quantification of multiple miRNAs in biological samples. Our experimental results showed a quantitative measurement of sequence-specific miRNAs with the detection limit in the femtomolar range, nearly 5 orders of magnitude lower than that of conventional molecular beacons. This amplification strategy also demonstrated a high selectivity for discriminating differences between miRNA family members. Considering the superior sensitivity and specificity, as well as the multiplex and simple-to-implement features, this method promises a great potential of becoming a routine tool for simultaneously quantitative analysis of multiple miRNAs in tissues or cells, and supplies valuable information for biomedical research and clinical early diagnosis.